Primate (particularly human) ES cell lines have widespread utility in connection with human developmental biology, drug discovery, drug testing, and transplantation medicine. For example, current knowledge of the post-implantation human embryo is largely based on a limited number of static histological sections. Because of ethical considerations the underlying mechanisms that control pluripotency, differentiation and developmental decisions of the early human embryo remain essentially unexplored.
Recently, however, primate (e.g. monkey and human) pluripotent embryonic stem cells have been derived from preimplantation embryos. See, for example, U.S. Pat. No. 5,843,780 and J. Thomson et al., 282 Science 1145-1147 (1998). The disclosure of these publications and of all other publications referred to herein are incorporated by reference as if fully set forth herein. Notwithstanding prolonged culture, these cells stably maintain a developmental potential to form advanced derivatives of all three embryonic germ layers.
It is generally known, however, that stem cells are defined to be cells which are capable both of self-renewal and differentiation into one or more differentiated cell types. Human embryonic stem cells are a category of stem cells created from human pre-implantation blastocysts. Human embryonic stem cells are pluripotent and may be totipotent, meaning that they can certainly differentiate into many cell types evidenced in an adult human body and may be capable of differentiating into all cell types present in the human body.
It is believed that one of the exciting potential uses of stem cells is for human tissue transplantation. It is hoped and expected that techniques can be developed to direct the differentiation of stem cells into specific lineages, which can then be transferred into the human body to replace or enhance tissues of the body. In order to do that, there first needs to be a clear understanding as to how cells become pluripotent in comparison to other cells. Next, techniques must be developed to direct the differentiation of stem cells into the specific cell lineages desired. Techniques have already been proposed to direct stem cell cultures into lineages of hematopoietic, neural, cardiomyocyte, pancreatic and other lineages. These techniques have proven to be quite different from each other and independent in the sense that a new and different technique is required for each new desired lineage.
Unfortunately, it still remains largely unknown why some cells become pluripotent and others do not. It is generally understood that in the early mammalian embryo, cleavage-stage blastomeres and at least some cells of the blastocyst's inner cell mass (ICM) all have the potential to contribute to any cell type of the body (Pedersen, 1986). ES cells, which are derived from early embryonic cells, can be expanded in vitro without limit, and retain the ability to form any cell type of the body (Evans and Kaufman, 1981; Martin, 1981; Thomson et al., 1998). Only a few key factors indicating pluripotency, such as Oct4 (Nichols et al., 1998) and Nanog (Chambers et al., 2003), have been identified so far, and the underlying mechanisms which control and maintain this remarkable state are largely yet unknown.
In contrast to the little that is known in the art about the control of pluripotency, there has been extensive characterization of the pathways controlling programmed cell death over the last three decades (Kerr et al., 1972; Ellis and Horvitz, 1986; Ellis et al., 1991). Programmed cell death and its morphological manifestation, apoptosis, are controlled by a complex, well-characterized genetic program in which mitochondria often have a central role (Ellis et al., 1991). During the course of programmed cell death, the mitochondrial membrane potential, ΨΨm, decreases, and the mitochondria release small proteins, including cytochrome c (Liu et al., 1996) and apoptosis inducing factor (AIF) (Green and Reed, 1998; Joza et al., 2001). This release ultimately results in the activation of some cysteine proteases, or caspases (Thomberry and Lazebnik, 1998). The caspases are divided into a group of initiator caspases (Earnshaw et al., 1999), including caspase-2, -8, -9, and -10, which promote programmed cell death in its early phases, and a group of terminal executioner caspases, including caspase-3, -6, and -7, which cleave several vital proteins, including poly(ADPribosyl) polymerase or PARP-1 (Lazebnik et al., 1994). Proteolysis of PARP-1 and other proteins eventually causes a sequential and controlled breakdown of the cell (Kidd, 1998).
Some studies have suggested that the programmed cell death system emerged concomitantly with the initial evolution of the metazoans (Aravind et al., 2001). Also, metazoans are believed to be the first multicellular animals having various types of cells organized into different types of tissues and organs. Therefore, it is of fundamental importance to understand what causes cells to specialize into different types of cells. While it has been demonstrated that human ES cells will differentiate into many progeny cells types, it has been difficult for researchers to create distinct and uniform cultures of progeny of human ES cells, which can be directed into a particular lineage or lineages. Accordingly, a need exists for the investigation of novel pathways and development of techniques that can be used to stably culture and direct primate embryonic stem cell differentiation into specific cell types as uniformly as practicable.